The recently characterized QueF class of enzymes reduce nitrites to primary amines. Enzymes that catalyze such reactions are referred to as nitrile oxidoreductases. QueF orthologs can be found in bacteria (such as Escherichia coli QueF and Bacilus subtilis QueF).(SEQ ID NO: 1)). As described in U.S. Pat. No. 7,364,882, which is incorporated herein by reference, QueF catalyzes the first known example of a biological conversion of a nitrile containing metabolite to its corresponding amine. More specifically, QueF catalyzes a late step reaction in the biosynthesis of the transfer RNA (tRNA)-modified nucleoside, queuosine (Q), a key modulator of ribosomal translational fidelity. (Van Lanen, J. S. et al. 2005; Reader, Metzgar et al. 2004.) Specifically, QueF catalyzes the nicotinamide adenine diphosphate (NADPH)-dependent, two-fold reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1), the advanced, and last common intermediate in the biosynthesis of Q. (Id.) Subsequent to the conversion of preQ0 to PreQ1, PreQ1 is inserted into the tRNA by the enzyme tRNA transglycosylase (TGT), and the remainder of the pathway occurs at the level of the tRNA. (Iwata-Reuyl 2003.)
Based on their amino acid sequences, QueF enzymes fall in two structural subfamilies (Van Lanen, J. S. et al. 2005). The YkvM subfamily is comprised of ˜160-amino add unimodular proteins with a characteristic QueF motif, i.e., E(S/L)K(S/A)hK(L/Y)(Y/F/W) (wherein h is a hydrophobic amino acid) bracketed on the N- and C-terminal sides by an invariant Cys and Glu, respectively. The YqcD subfamily of QueF enzymes is characterized by ˜280-amino acid bimodular proteins where the QueF motif and the invariant Cys and Glu are located separately, in the weakly homologous N- and C-terminal halves (modules) of the polypeptide chain, respectively. Functional analysis of an enzyme from each subfamily, YkvM (B. subtilis QueF) and YqcD (E. coli QueF), showed that YqcD enzymes are homodimers while YkvM enzymes function as higher order multimers.
The crystal structure of YkvM unimodular QueF complexed with preQ0 reveals an asymmetric tunnel-fold homodecamer of two head-to-head facing pentameric subunits cyclically arranged to form a 20-stranded β-barrel, layered on the outside by 10α-helices, an architecture characteristic of unimodular pterin and purine binding enzymes. The structure harbors 10 active sites each located at the interface between three monomers. Eight active sites are each occupied with a preQ0 molecule that is anchored by the invariant Glu98. The preQ0 molecule also forms a covalent adduct with the catalytic residue, Cys55. The empty sites are associated with two subunits that are slightly off the 5-fold symmetry axis, and exhibit disordered C-terminal regions. A glucose-6-sulfate (G6S) or glucosamine moiety, originating from dextran sulfate, occupies the previously predicted NADP site comprised of residues from two subunits and includes residues from the conserved QueF motif E79(S/L)K(S/A)hK(L/Y)(Y/F/W)86. Based on the foregoing structural characterization of QueF, native QueF enzymes can be mutated to engineer other nitrile oxido-reductases which have specificities for other nitile containing substrates. Engineered QueF enzymes can be used, for example, in methods that provide a nitrile oxido-reductase (such as a recombinant nitrile oxido-reductase) and contacting the nitrile containing compound with the nitrile oxido-reductase under conditions sufficient for substantially reducing the nitrile containing compound to the corresponding amine. Such methods can be performed either in vitro or in vivo.
The discovery of QueF activity expands the chemistry of known nitrile metabolizing enzymes (Banerjee, Sharma et al. 2002), which includes hydrolysis (nitrile hydratase and nitrilase), oxidation (oxygenase), and cleavage (hydroxynitrile lyase). Prior to the discovery of QueF activity, the reduction of a nitrile was unprecedented in biology. Until then, industrial processes that relied on nitrile reduction had to resort to non-biological methods of reducing nitriles. Traditionally, the reduction of nitrites to amines has been carried out synthetically by hydrogenation over various transition metal catalysts or by metal hydride reductions. However, those reactions are typically non-selective; and thus, require the use of protecting groups when other reducible functional groups are present, and result in the formation of unwanted byproducts. Conversely, methods that utilize QueF as a biocatalyst in the transformation of nitriles to their corresponding amities can provide an environmentally sensitive alternative to the synthetic conversion of nitrites to amines. To that end, the present invention is directed to the crystal structure of B. subtilis QueF (19.4 kDa, 165 amino acids (SEQ ID NO: 1)). The crystal structure and structural data on the active site architecture and substrate and cofactor binding pockets of QueF may be used for the design and development of QueF mutants that bind to a variety of nitrite containing, industrially important substrates and catalyze the reduction of nitriles to their corresponding amines.